Transcriptional regulation of cyclin-dependent kinase inhibitor 1A (P21) by the transcription factor AP-2γ

AP-2 transcription factors constitute a family of sequence-specific DNA-binding proteins encoded by five highly homologous yet functionally distinct genes, AP-2α to AP-2ε. AP-2γ appears to play a major role in breast cancer, being expressed in a large proportion of primary tumours. In this study we have analysed in more detail the mechanism of transcriptional regulation of the p21/cyclin-dependent kinase inhibitor 1A (p21/CDKN1A) gene by AP-2γ.

Silencing of AP-2γ was carried out in MCF-7 cells using siRNA or doxycycline inducible shRNA. Chromatin immunoprecipitation (ChIP) assays were performed using specific antibodies against AP-2γ (H77), AP-2α, Myc, histone demethylase PLU1/JARID1B, histone H3 and trimethyl dimethyl and monomethyl histone H3 followed by quantitative PCR. Electrophoretic mobility shift assay (EMSA) competition assay and reporter assays were used to identify the AP-2 binding site.

Silencing of AP-2γ by either siRNA or inducible shRNA inhibits cell proliferation and results in upregulation of p21/CDKN1A expression with no induction of apoptosis. ChIP assays demonstrated binding of AP-2γ, PLU1/JARID1B and Myc to a region adjacent to the transcription start site of the p21/CDKN1A gene. Reduction in the binding of cMyc and PLU1/JARID1B and increased levels of histone H3 trimethyl-K4 were observed at the proximal region of p21/CDKN1A promoter after silencing of AP-2γ. Treatment of MCF-7 cells with the antimitotic drug vinblastine but not with hydroxyurea reduced the CDKN1A binding of AP-2γ, PLU-1/JARID1B and Myc. H3396 cells treated with the oestrogen receptor inhibitor Faslodex, which upregulates p21/CDKN1A, decreased AP-2γ binding but increased binding of AP-2α at the p21/CDKN1A promoter. EMSA competition assays and reporter assays showed that AP-2γ and AP-2α bind to a new site (GCC N3 GGG) at position -105 of the p21/CDKN1A promoter.

The repression of the p21/CDKN1A gene by AP-2γ may contribute to the activation of proliferation associated with this transcription factor in breast cancer.